In 1558, the DauphinFrancis married Mary, Queen of Scots. When the sickly young man became king after his father's death in 1559, the queen's uncles, the Duke of Guise and his brother the Cardinal of Lorraine, controlled French politics during his short reign.

The House of Guise claimed descent from Charlemagne, and harbored pretensions to the French crown. In the reign of Francis II they attained supreme power, and sought to convert it to true kingship by eradicating the House of Bourbon, the legal successors to the throne of France. The leading Bourbon princes, Antoine, King of Navarre and Louis, Prince of Conde, were Protestants. To oppose the Bourbons, the Guises made themselves champions of the Catholic faith and allied themselves with the Philip II of Spain. The persecution of Protestants increased in vigor.

This prompted the Amboise conspiracy, in which the Huguenots and the Bourbons plotted to overthrow the power of the House of Guise. The Guise family brutally put down the conspiracy. Near the end of the year both the King of Navarre and the Prince of Condé were in their hands—one destined for assassination and the other for execution—when the death of the king, Francis II, prevented the fulfillment of their plans. After King Francis' death they opposed the more tolerant policy of the Regent, Catherine de' Medici, and their actions provoked the French Wars of Religion.

The Duke Francis helped to defeat the Huguenots at the Battle of Dreux, but he was assassinated shortly afterward, in 1563. His son, Henry of Guise, became the third Duke of Guise (1550–1588). He helped plan the infamous St. Bartholomew's Day Massacre and was responsible for the formation of the Catholic League. The death of the royal heir-presumptive, the Francis, Duke of Anjou, in 1584, which made the Protestant King Henry of Navarre heir to the French throne, led to a new civil war, the War of the Three Henries, with King Henry III of France, Henry of Navarre, and Henry of Guise all fighting for control of France. Guise began the war by declaring the unacceptability of Navarre as King of France, and his control of the powerful Catholic League soon forced the French king to follow in his wake. Immensely ambitious, in 1588 Guise, with Spanish support, instigated a revolt against the king, taking control of the city of Paris and becoming the de facto ruler.

The Duke of Mayenne's nephew, the young Duke of Guise, Charles, was proposed by the Catholic League as a candidate for the throne, possibly through a marriage to Philip II of Spain's daughter Isabella, the granddaughter of Henry II of France. The Catholic League was eventually defeated, but for the sake of the country King Henry IV bought peace with Mayenne, and in January 1596 a treaty was signed that put an end to the League.

After this, the House of Guise receded from its prominent position in French politics, and the senior line, that of the Dukes of Guise became extinct in 1688. The vast estates and title were disputed and diverted by various relatives, although several junior branches of the family (Dukes of Mayenne, Dukes of Elbeuf, etc.) perpetuated the male line until 1825. Thereafter, the only surviving male branch of the House of Lorraine was the seniormost branch, which had exchanged the sovereign duchy of Lorraine for that of the Grand Duchy of Tuscany, holding sovereignty as the Habsburg-Lorraine Emperors of Austria-Hungary into the 20th century.